Energy balance (involving energy metabolism, nutritional state, lipid storage and the like) is an important criteria for health. This energy homeostasis involves food intake and metabolism of carbohydrates and lipids to generate energy necessary for voluntary and involuntary functions. Metabolism of proteins can lead to energy generation, but preferably leads to muscle formation or repair. Among other consequences, a lack of energy homeostasis lead to over or under formation of adipose tissue.
Formation and storage of fat is insulinmodulated. For example, insulin stimulates the transport of glucose into cells, where it is metabolized into xcex1-glycerophosphate which is used in the esterification of fatty acids to permit storage thereof as triglycerides. In addition, adipocytes (fat cells) express a specific transport protein that enhances the transfer of free fatty acids into adipocytes.
Adipocytes also secrete several proteins believed to modulate homeostatic control of glucose and lipid metabolism. These additional adipocyte-secreted proteins include adipsin, complement factors C3 and B, tumor necrosis factor xcex1, the ob gene product and Acrp30. Evidence also exists suggesting the existence of an insulin-regulated secretory pathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995. Over or under secretion of these moieties, impacted in part by over or under formation of adipose tissue, can lead to pathological conditions associated directly or indirectly with obesity or anorexia.
Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a amino-terminal signal sequence, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain. See, Scherer et al. as described above and International Patent Application No. WO96/39429. Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the carboxy-terminal globular domain, to complement factor Clq and to a summer serum protein of hibernating Siberian chipmunks (Hib27). Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.
Another secreted protein that appears to be exclusively produced in adipocytes is apM1, described, for example, in Maeda et al., Biochem. Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 amino acid open reading frame and a long 3xe2x80x2 untranslated region. The protein included a signal sequence, an amino-terminal non-collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, collagen VIII and complement protein C1q.
Complement factor C1q consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region. Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks. A globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain. Clq is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain.
Molecules capable of modulating energy homeostasis are sought for the study of this phenomena and for the prevention or treatment of imbalances. Also, molecules capable of modulating adipocyte secretory pathways are also sought as indirect energy homeostasis modulators and as research reagents.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect of the invention is provided an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO:2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO:2. Within one embodiment the polypeptide is at least 90% identical to SEQ ID NO:2. Within another embodiment the polypeptide comprises a collagen-like domain having at least 22 collagen repeats. Within another embodiment the polypeptide comprises residues 19-243 of SEQ ID NO:2. Within yet another embodiment the polypeptide is covalently linked amino terminally or carboxy terminally to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.
Within another aspect is provided an isolated polypeptide selected from the group consisting of: a) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 95 of SEQ ID NO:2; b) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 96 of SEQ ID NO:2; and c) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to 97 of SEQ ID NO:2; d) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 98 of SEQ ID NO:2; e) a polypeptide having a sequence of amino acid residues from amino acid residue 98 to amino acid residue 243 of SEQ ID NO:2; f) a polypeptide having a sequence of amino acid residues from amino acid residue 99 to amino acid residue 243 of SEQ ID NO:2; g) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 243 of SEQ ID NO:2; and h) a polypeptide having a sequence of amino acid residues that is 90% identical in amino acid sequence to a), b), c), d), e), f), g) or h).
Within another aspect is provided a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, the first portion comprising a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO:2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2 containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2, containing the globular-like domain or the receptor binding portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2, including the collagen-like domain and the globular domain; and the second portion comprising another polypeptide. Within one embodiment the first portion is selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 30 to amino acid residue 95 of SEQ ID NO:2; b) a polypeptide having the sequence of amino acid residue 30 to amino acid residue 96 of SEQ ID NO:2; c) a polypeptide having the sequence of amino acid residue 30 to amino acid residue 97 of SEQ ID NO:2; d) a polypeptide having the sequence of amino acid residue 30 to amino acid residue 98 of SEQ ID NO:2; e) a polypeptide having the sequence of amino acid residue 30 to amino acid residue 243 of SEQ ID NO:2; f) a polypeptide having the sequence of amino acid residue 98 to amino acid residue 243 of SEQ ID NO:2; and g) a pplypeptide having the sequence of amino acid residue 99 to amino acid residue 243 of SEQ ID NO:2.
Within another aspect is provided a fusion protein comprising a secretory signal sequence having the 35 amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ ID NO:2, wherein the secretory signal sequence is operably linked to an additional polypeptide.
Within yet another aspect is pharmaceutical composition comprising a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.
Also provided is an antibody that specifically binds to an epitope of a polypeptide as described above.
Further provided is an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO:2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO:2. Within one embodiment the polypeptide is at least 90% identical to SEQ ID NO:2. Within another embodiment the polypeptide comprises a collagen-like domain having at least 22 collagen repeats. Within another embodiment the polynucleotide is DNA.
Within yet another aspect is provided an isolated polynucleotide selected from the group consisting of: a) a sequence of nucleotides from nucleotide 243 to nucleotide 962 of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 252 to nucleotide 962 of SEQ ID NO:1; c) a sequence of nucleotides from nucleotide 285 to nucleotide 482 of SEQ ID NO:1; d) a sequence of nucleotides from nucleotide 285 to nucleotide 485 of SEQ ID NO:1; e) a sequence of nucleotides from nucleotide 285 to nucleotide 488 of SEQ ID NO:1; f) a sequence of nucleotides from nucleotide 285 to nucleotide 491 of SEQ ID NO:1; g) a sequence of nucleotides from nucleotide 285 to nucleotide 926 of SEQ ID NO:1; h) a sequence of nucleotides from nucleotide 491 to nucleotide 926 of SEQ ID NO:1; i) a polynucleotide encoding a polypeptide having sequence of nucleotides that is at least 80% identical n nucleotide sequence to a), b), c), d), e), f), g) and h); j) nucleotide sequences complementary to a), b), c), d), e), f), g), h) or i); and k) degenerate nucleotide sequences of a), b), c), d), e), f), g), h), i) or j).
Within another aspect is provided an isolated polynucleotide encoding a fusion protein. consisting essentially of a first portion and a second portion joined by a peptide bond, the first portion is selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO:2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO:2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2 containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2, containing the globular-like domain or an active portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO:2, including the collagen-like domain and the globular domain; and the second portion comprising another polypeptide.
Within another aspect is provided an isolated polynucleotide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ ID NO:2, wherein the secretory signal sequence is operably linked to an additional polypeptide.
Within yet another aspect is an isolated polynucleotide comprising the sequence of nucleotide 1 to nucleotide 729 of SEQ ID NO:10.
Also provided is an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator. Within one embodiment the DNA segment encodes a polypeptide that is at least 90% identical to SEQ ID NO:2. Within another embodiment the DNA segment encodes a polypeptide further comprising a collagen-like domain having at least 22 collagen repeats. Within yet another embodiment the DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag. Within still another embodiment the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide. Within yet another embodiment the secretory signal sequence comprises residues 1-15 or 1-18 of SEQ ID NO:2.
Also provided is a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator; wherein the cell expresses the polypeptide encoded by the DNA segment.
Within another aspect is provided a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide as described above; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.
Within another aspect is an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:10 or a sequence complementary to SEQ ID NO:10.
Within yet another aspect is a method for modulating free fatty acid metabolism by administering a pharmaceutically effective dose of a polypeptide as described above.